Bottom Line:
This effect coincided with enhanced phosphorylation of the Na+-K+-2Cl- cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl- levels.In contrast, the mRNA levels of the two transporters did not change markedly during this time.Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Background: Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl- accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl- currents. The intracellular Cl- concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl- currents to signal generation differs between individual afferent neurons, and whether the specific Cl- levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl- homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl- concentrations and on the expression levels of Cl- transporters in rat DRG neurons.

Results: We developed an in vitro assay for testing how inflammatory mediators influence Cl- concentration and the expression of Cl- transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 microM ATP, 0.9 microM bradykinin, and 1.4 microM PGE2 for 1-3 hours. Two-photon fluorescence lifetime imaging with the Cl--sensitive dye MQAE revealed an increase of the intracellular Cl- concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na+-K+-2Cl- cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl- levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl- importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl- homeostasis toward a state of augmented Cl- accumulation, which is induced by a 1-3 hour treatment with inflammatory mediators.

Conclusion: Our findings indicate that inflammatory mediators impact on Cl- homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl- levels and, hence, the driving force for depolarizing Cl- efflux. These findings corroborate current concepts for the role of Cl- regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl- concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl- currents in the presynaptic terminals. Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Figure 1: In vitro assay to study the effects of inflammatory mediators by immunohistochemistry. (A) Increased expression of neuropeptides during treatment with inflammatory mediators. The immunfluorescence of calcitonin gene-related peptide (CGRP) and substance P (SP) was evaluated at the indicated times after start of the treatment. The relative number of neuropeptide-expressing neurons was determined in test DRGs and in contralateral control DRGs, and the percent change of positive cells, Δ NP = (NPtest-NPctr)/NPctr·100 [%], is given for each time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**). (B) Cryosections of a control DRG and the treated contralateral test DRG stained with NKCC1 antibodies. The red circles illustrate the area from which the mean fluorescence intensity was obtained. Background subtraction removed most of the pericellular fluorescence as well as the fluorescence originating from the nucleus. All cell sections with intact perimeter were evaluated. (C) Distribution of fluorescence intensities recorded from 3 control DRGs (blue) and 3 contralateral test DRGs (red) illustrate a shift to higher intensities after a 3-hr treatment with inflammatory mediators. Solid lines are Gaussian fits to the data.

Mentions:
Our objective was to monitor changes of the intracellular Cl- concentration as well as changes in expression of NKCC1 and KCC2 in the presence of inflammatory mediators. To minimize damage to the neurons and to avoid dedifferentiation effects that may alter Cl- homeostasis in cell culture, we examined the neurons in rat DRGs with intact dura mater. The DRGs were excised and immediately used for the assay which was concluded within < 4 hr post mortem. The excised DRGs were incubated with a combination of inflammatory mediators (100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, 1.4 μM PGE2) for 1, 2, or 3 hr at 37°C, and then either subjected to 2P-FLIM analysis or fixed for immunohistochemistry. The vitality of DRG neurons during this procedure was tested in DRG tissue slices using propidium-iodide exclusion and resazurin-reduction assays (see Methods). Both tests confirmed a cell viability of > 95% within the 3-hr period of experimentation with and without the inflammatory mediators (not shown). To find out whether the DRG neurons responded to the inflammatory mediators, we analyzed the immunosignals of the neuropeptides CGRP and substance P (SP) which are known to be upregulated during inflammation [e.g. [36-38]]. We compared the relative numbers of neuropeptide-expressing neurons in DRGs treated with inflammatory mediators to control DRGs from the same animal treated with exactly the same protocol, but without the inflammatory mediators. In 9 control DRGs (T7-T9) from 3 animals, we found on average 35% of the neurons expressing CGRP and 33% expressing SP. The 9 corresponding contralateral test DRGs were treated with inflammatory mediators for 1 hr (3 T7-DRGs from 3 rats), 2 hr (3 T8-DRGs), or 3 hr (3 T9-DRGs). We determined the relative number of neuropeptide-positive cells separately for each control DRG (NPctr) and for its contralateral test DRG (NPtest), and we quantified the percent change of neuropeptide-positive neurons according to Δ NP = (NPtest-NPctr)/NPctr·100 [%]. Fig. 1A illustrates the time-dependent increase of neuropeptide-positive neurons in the treated ganglia. Δ NP (± SD; 3 animals) was 4 ± 2% (1 hr), 10 ± 4% (2 hr), and 29 ± 5% (3 hr) for CGRP, and 6 ± 7% (1 hr), 18 ± 5% (2 hr), and 34 ± 10% (3 hr) for SP. In total, 5329 control cells and 4596 test cells were counted for the CGRP statistics, and 3178 control cells and 3467 treated cells for SP. These data show that the DRG neurons in our in vitro assay respond to the inflammatory mediators with enhanced synthesis of neuropeptides, a behaviour which is indicative of an inflammatory response in vivo. To monitor changes of [Cl-]i between control and treated DRGs we examined the Cl--dependent fluorescence of MQAE by 2P-FLIM immediately after the test time of 1, 2, or 3 hours, as described below.

Figure 1: In vitro assay to study the effects of inflammatory mediators by immunohistochemistry. (A) Increased expression of neuropeptides during treatment with inflammatory mediators. The immunfluorescence of calcitonin gene-related peptide (CGRP) and substance P (SP) was evaluated at the indicated times after start of the treatment. The relative number of neuropeptide-expressing neurons was determined in test DRGs and in contralateral control DRGs, and the percent change of positive cells, Δ NP = (NPtest-NPctr)/NPctr·100 [%], is given for each time. Indicated significance levels are p ≤ 0.05 (*) or p ≤ 0.01 (**). (B) Cryosections of a control DRG and the treated contralateral test DRG stained with NKCC1 antibodies. The red circles illustrate the area from which the mean fluorescence intensity was obtained. Background subtraction removed most of the pericellular fluorescence as well as the fluorescence originating from the nucleus. All cell sections with intact perimeter were evaluated. (C) Distribution of fluorescence intensities recorded from 3 control DRGs (blue) and 3 contralateral test DRGs (red) illustrate a shift to higher intensities after a 3-hr treatment with inflammatory mediators. Solid lines are Gaussian fits to the data.

Mentions:
Our objective was to monitor changes of the intracellular Cl- concentration as well as changes in expression of NKCC1 and KCC2 in the presence of inflammatory mediators. To minimize damage to the neurons and to avoid dedifferentiation effects that may alter Cl- homeostasis in cell culture, we examined the neurons in rat DRGs with intact dura mater. The DRGs were excised and immediately used for the assay which was concluded within < 4 hr post mortem. The excised DRGs were incubated with a combination of inflammatory mediators (100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, 1.4 μM PGE2) for 1, 2, or 3 hr at 37°C, and then either subjected to 2P-FLIM analysis or fixed for immunohistochemistry. The vitality of DRG neurons during this procedure was tested in DRG tissue slices using propidium-iodide exclusion and resazurin-reduction assays (see Methods). Both tests confirmed a cell viability of > 95% within the 3-hr period of experimentation with and without the inflammatory mediators (not shown). To find out whether the DRG neurons responded to the inflammatory mediators, we analyzed the immunosignals of the neuropeptides CGRP and substance P (SP) which are known to be upregulated during inflammation [e.g. [36-38]]. We compared the relative numbers of neuropeptide-expressing neurons in DRGs treated with inflammatory mediators to control DRGs from the same animal treated with exactly the same protocol, but without the inflammatory mediators. In 9 control DRGs (T7-T9) from 3 animals, we found on average 35% of the neurons expressing CGRP and 33% expressing SP. The 9 corresponding contralateral test DRGs were treated with inflammatory mediators for 1 hr (3 T7-DRGs from 3 rats), 2 hr (3 T8-DRGs), or 3 hr (3 T9-DRGs). We determined the relative number of neuropeptide-positive cells separately for each control DRG (NPctr) and for its contralateral test DRG (NPtest), and we quantified the percent change of neuropeptide-positive neurons according to Δ NP = (NPtest-NPctr)/NPctr·100 [%]. Fig. 1A illustrates the time-dependent increase of neuropeptide-positive neurons in the treated ganglia. Δ NP (± SD; 3 animals) was 4 ± 2% (1 hr), 10 ± 4% (2 hr), and 29 ± 5% (3 hr) for CGRP, and 6 ± 7% (1 hr), 18 ± 5% (2 hr), and 34 ± 10% (3 hr) for SP. In total, 5329 control cells and 4596 test cells were counted for the CGRP statistics, and 3178 control cells and 3467 treated cells for SP. These data show that the DRG neurons in our in vitro assay respond to the inflammatory mediators with enhanced synthesis of neuropeptides, a behaviour which is indicative of an inflammatory response in vivo. To monitor changes of [Cl-]i between control and treated DRGs we examined the Cl--dependent fluorescence of MQAE by 2P-FLIM immediately after the test time of 1, 2, or 3 hours, as described below.

Bottom Line:
This effect coincided with enhanced phosphorylation of the Na+-K+-2Cl- cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl- levels.In contrast, the mRNA levels of the two transporters did not change markedly during this time.Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.

Background: Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl- accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl- currents. The intracellular Cl- concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl- currents to signal generation differs between individual afferent neurons, and whether the specific Cl- levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl- homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl- concentrations and on the expression levels of Cl- transporters in rat DRG neurons.

Results: We developed an in vitro assay for testing how inflammatory mediators influence Cl- concentration and the expression of Cl- transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 microM ATP, 0.9 microM bradykinin, and 1.4 microM PGE2 for 1-3 hours. Two-photon fluorescence lifetime imaging with the Cl--sensitive dye MQAE revealed an increase of the intracellular Cl- concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na+-K+-2Cl- cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl- levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl- importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl- homeostasis toward a state of augmented Cl- accumulation, which is induced by a 1-3 hour treatment with inflammatory mediators.

Conclusion: Our findings indicate that inflammatory mediators impact on Cl- homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl- levels and, hence, the driving force for depolarizing Cl- efflux. These findings corroborate current concepts for the role of Cl- regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl- concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl- currents in the presynaptic terminals. Moreover, excitatory Cl- currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.